Identification of a cDNA Coding for the SerH3 Surface Protein of Tetrahymena thermophila

We have identified a Tetrahymena thermophila cDNA-containing plasmid (pC6) which hybridizes to a 1.47-kB RNA whose changes in cellular concentration parallel the changes in synthetic rate of a major cell surface protein. From a molecular and genetic analysis of strains expressing the gene ( SerH3 ) encoding this protein, and of strains expressing immunologically distinct alleles of this gene, we conclude that pC6 encodes a portion of the SerH3 allele.     

Isolation and Genetic Analysis of Mutations at the SerH Immobilization Antigen Locus of TETRAHYMENA THERMOPHILA

Multiple alleles at the SerH locus specify the major cell surface protein (immobilization antigen) of the ciliate Tetrahymena thermophila. Following mutagenesis of SerH1 homozygotes, two mutations, H1-1 and H1-2, were recovered in heterozygous form. Mutant homozygotes do not express H1 antigen, nor is H1 expressed in F1 progeny of crosses to wild-type strains homozygous for SerH2 or SerH3. H1-1 and H1-2 segregate without recombination from these wild-type alleles in expected F2 and testcross Mendelian ratios. H1-1 and H1-2 do, however, complement each other to express H1 antigen. Experiments suggest this complementation is due neither to recombination during macronuclear development nor to interallelic complementation of defective SerH1 gene products. These results suggest that SerH1 is intact in one mutant, and possibly both, although no such allele has been segregated in testcross progeny (N = 205). The hypothesis is presented that complementation between H1-1 and H1-2 is due to interaction between allele-specific regulators closely linked to the SerH1 gene.     

Evidence for the requirement of protein synthesis and protein kinase activity in the temperature regulated stability of a Tetrahymena surface protein mRNA.

In Tetrahymena thermophila, the expression of the temperature-specific surface protein SerH3 is controlled primarily by a temperature-dependent change in the stability of its mRNA. The change in SerH3 mRNA stability occurs very rapidly after a shift in incubation temperature. This change in temperature could affect SerH3 mRNA stability directly by producing structural changes in the mRNA or regulatory factors acting on SerH3 mRNA. Alternatively, the temperature change could act indirectly through a signal transduction pathway leading to de novo synthesis of new regulatory factors or modifications of existing regulatory factors. To address these issues, we monitored the effect of temperature on an in vitro SerH3 mRNA decay assay and the in vivo effects of a variety of inhibitors against protein synthesis and protein kinases on SerH3 mRNA stability. The results of Northern analysis of SerH3 mRNAs in an in vitro mRNA decay assay indicate that temperature alone can not change the half-life of this mRNA. Furthermore, slot blot analysis of cytoplasmic RNAs show that protein synthesis and the action of protein kinases are not required for SerH3 mRNA turnover in cells grown at 30 degrees C. In contrast, our results indicate that the rapid decay of the SerH3 mRNA in cells grown at 30 degrees C and shifted to 40 degrees C requires a one time serine/threonine phosphorylation event which occurs at the temperature shift. In addition, the data show that a regulatory protein involved in rapid SerH3 mRNA decay must be newly and continuously synthesized following the temperature shift from 30 to 40 degrees C. These data show the complexity of temperature regulated mRNA decay and indicate that phosphorylation and protein synthesis are major factors in this process.     

A Temperature-Sensitive Mutation of the Temperature-Regulated Serh3 I-Antigen Gene of Tetrahymena Thermophila: Implications for Regulation of Mutual Exclusion

The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36°. In homozygotes grown at the nonpermissive temperature (>32°), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36° cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sequencing of SerH3-ts1 revealed a single A -> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein.     

Reproducible and variable rearrangements of a Tetrahymena thermophila surface protein gene family occur during macronuclear development.

The expression of Tetrahymena surface proteins serotype H3 (SerH3) and serotype T (SerT) is under environmental regulation. SerH3 is expressed when cells are incubated between the temperatures of 20 and 35 degrees C, while SerT is expressed when cells are grown at temperatures above 35 degrees C. Using a SerH3 cDNA clone as a hybridization probe, we determined that (i) the SerH3 gene is a member of a multigene family; (ii) most members of this multigene family are variably rearranged during macronuclear development; and (iii) the gene which produces the SerH3 mRNA is reproducibly rearranged during macronuclear development.     

Heterogeneity of chromosomal genes encoding chloramphenicol resistance in streptococci

The DNA of 21 chloramphenicol-resistant plasmid-free streptococci was tested for sequence homology with the genes encoding chloramphenicol acetyltransferase (cat) of the staphylococcal plasmids pC194 and pC221. Homology to the cat gene of pC194 was detected in 11 strains, including the 8 strains of Streptococcus pneumoniae examined, and homology to cat of pC221 was found in 3 strains. The DNA of 7 strains did not detectably hybridize with either probe.     

Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins.

We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors.     

Immobilization Antigens from Tetrahymena thermophila Are Glycosyl-Phosphatidylinositol-Linked Proteins

We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [³H]ethanolamine or [³H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [³H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [³H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors.     

Polymorphism and selection at the SerH immobilization antigen locus in natural populations of Tetrahymena thermophila

The SerH locus of Tetrahymena thermophila is one of several paralogous loci with genes encoding variants of the major cell surface protein known as the immobilization antigen (i-ag). The locus is highly polymorphic, raising questions concerning functional equivalency and selective forces acting on its multiple alleles. Here, we compare the sequences and expression of SerH1, SerH3, SerH4, SerH5, and SerH6. The precursor i-ags are highly similar. They are rich in alanine, serine, threonine, and cysteine and they share nearly identical ER translocation and GPI addition signals. The locations of the 39 cysteines are highly conserved, particularly in the 3.5 central, imperfect tandem repeats in which 8 periodic cysteines punctuate alternating short and long stretches of amino acids. Hydrophobicity patterns are also conserved. Nevertheless, amino acid sequence identity is low, ranging from 60.7 to 82.9%. At the nucleotide level, from 9.7 to 26.7% of nucleotide sites are polymorphic in pairwise comparisons. Expression of each allele is regulated by temperature-sensitive mRNA stability. H mRNAs are stable at <36 degrees but are unstable at >36 degrees. The H5 mRNA, which is less affected by temperature, has a different arrangement of the putative mRNA destabilization motif AUUUA. Statistical analysis of SerH genes indicates that the multiple alleles are neutral. Significantly low ratios of the rates of nonsynonymous to synonymous amino acid substitutions suggest that the multiple alleles are subject to purifying (negative) selection enforcing constraints on structure.     

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.     

Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.     

A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid

Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.     

Multiple Effects of Mutation on Expression of Alternative Cell Surface Protein Genes in Tetrahymena Thermophila

Genes at the SerH locus of the ciliated protist Tetrahymena thermophila specify the major (H) surface protein on cells grown at 20-36 degrees. Alternative proteins L, T, S and I are expressed under different conditions of temperature and culture media. Mutants unable to express SerH genes were examined for expression of these proteins, also called immobilization or i-antigens, at both H and non-H conditions. In all instances, one or more i-antigens were expressed in the absence of H, and, in most instances, expression of i-antigens under non-H conditions was also affected. Examples of the latter include both the continued expression of H-replacement antigens and the inability to express certain other i-antigens. Such multiple effects were observed in mutants with trans-acting (rseA, rseB, rseC, RseD) and cis-acting (H1-1 and H1-2) mutations, but not in mutants in which SerH is affected developmentally (B2092, B2101, B2103, B2107). These interactions suggest that the wild-type genes identified by mutation exert both positive and negative effects in the regulation of i-antigen gene expression.     

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.     

The Third Replicon of Members of the Burkholderia cepacia Complex,Plasmid pC3, Plays a Role in Stress Tolerance

The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.     

水稻转录因子OsBP-73靶基因的初步筛选

OsBP-73是用酵母单杂交系统,以水稻蜡质基因(Wx)的顺式作用元件为诱饵,从水稻cDNA表达文库中筛选获得的转录因子。本文以OsBP-73基因为例介绍了用"下拉"(pull-down)方法筛选转录因子靶基因的一般步骤。将含有该基因DNA结合功能域的cDNA片段构建到原核表达载体上,并在大肠杆菌中诱导表达获得其蛋白p73。用纯化后的p73蛋白通过"下拉"实验对OsBP-73靶基因进行初步筛选,获得了22个阳性克隆,为进一步研究转录因子OsBP-73参与的水稻转录调控网络提供依据。     

禽流感病毒HA基因真核表达质粒的构建与表达

血凝素蛋白（HA）基因是禽流感病毒（AIV）重要的保护性抗原基因.为了研究 HA基因疫苗,用PCR扩增H5亚型AIV HA基因,将其克隆到质粒pcDNA4/HisMax和pRc/CMV上得到真核表达质粒pC4H5和pCMVH5.采用Tfx^TM-20、Superfect转染试剂和电转染法转染HeLa细胞,转染后的HeLa细胞经蛋白质印迹和血凝试验检测HA蛋白及其活性.结果表明,Superfect转染和电转染均能正确表达HA蛋白并具有生物学活性,蛋白质印迹检测到HA和HA裂解的HA1和HA2,与AIV 的HA、HA1、HA2蛋白的分子质量一致.从血凝试验结果看,Superfect和电转染表达的HA均具有血凝活性,而经Superfect转染的pC4H5的表达量是pCMVH5的8倍,表明pC4H5是一高效的真核表达质粒.